CDR grafted type III anti-CEA humanized mouse monoclonal antibodies

ABSTRACT

A Class III, anti-CEA monoclonal antibody, preferably a monoclonal antibody comprising the complementarity-determining regions (CDRs) of a parental murine Class III, anti-CEA monoclonal antibody is disclosed. This monoclonal antibody is preferably a humanized monoclonal antibody in which the CDRs are engrafted to the framework regions of a heterologous antibody, wherein the humanized monoclonal antibody retains the binding specificity of the parental murine monoclonal antibody. A preferred Class III, anti-CEA monoclonal antibody is one in which the preferred heterologous antibody is from a human. Also provided are DNA constructs, vectors, cells and methods for producing the Class III, anti-CEA monoclonal antibodies, and diagnostic and therapeutic conjugates using same. Methods of treatment with a Class III, anti-CEA monoclonal antibody and with the conjugate containing this monoclonal antibody are provided as well as methods of diagnosis with the conjugate containing this monoclonal antibody.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of application Ser. No. 09/253,794,filed Feb. 22, 1999, now pending, which is a divisional of applicationSer. No. 08/318,157, filed Oct. 5, 1994, now U.S. Pat. No. 5,874,540,which are both incorporated by reference in their entirety. Thisapplication claims only subject matter disclosed in the parentapplications and therefore presents no new matter.

BACKGROUND OF THE INVENTION

The invention relates to immunological reagents for diagnostic andtherapeutic use in colon and other cancers. In particular, the inventionrelates to humanized anti-carcinoembryonic antigen (“CEA”) monoclonalantibodies (“mAbs”) that have the binding affinity characteristics ofcorresponding mouse anti-CEA mAb (MN14) and the antigenic and effectorproperties of a human antibody. Further, the invention relates tohumanized mAbs in which the complementarity determining regions (“CDRs”)of an anti-CEA murine mAb is grafted into the framework regions of ahuman antibody, to DNAs that encode such CDR-grafted antibodies, tovectors and transformed hosts for propagating and expressing the DNAs,and to conjugates of the antibodies useful in diagnostic and therapeuticapplications.

A promising approach to cancer diagnosis and therapy involves the use oftargeting antibodies to deliver diagnostic and therapeutic agentsdirectly to the site of a malignancy. Over the past decade, a widevariety of tumor-specific antibodies and antibody fragments have beendeveloped, as have methods to conjugate the antibodies to drugs, toxins,radionuclides or other agents, and to administer the conjugates topatients. These efforts have produced great progress, but a variety oflargely unanticipated problems have limited the diagnostic andtherapeutic utility of some of the reagents thus far developed.

Among the most intractable problems is that which is caused by the humanimmune system itself, which may respond to the targeting conjugate as aforeign antigen. For instance, patients treated with drugs orradionuclides complexed with murine monoclonal antibodies (which havebeen the most commonly used targeting antibodies for human) developcirculating human anti-mouse antibodies (HAMAs) and a generalizedimmediate type-III hypersensitivity reaction to the antibody moiety ofthe conjugate. Furthermore, even when adverse side effects are minimal(for example, as in a single administration), circulating HAMAs decreasethe effective concentration of the targeting agent in the patient andtherefore limiting the diagnostic or therapeutic agent from reaching thetarget site.

Several approaches have been developed to overcome or avoid thisproblem, with only limited success. One strategy has been to chemicallymodify the targeting antibody to suppress its antigenicity. For example,conjugation of polyethylene glycol to the targeting antibody(PEGylation) is reported to reduce antigenicity of antibodies. Anotherapproach has been to characterize the situs of antigenicity in anantibody and then remove it. In this vein, Fab′, F(ab)₂ and otherantibody fragments have been used in place of whole IgG. In addition,attempts have been made to reduce the adverse effects of HAMA byplasmaphoretically removing HAMA from blood. Immunosuppressivetechniques also have been used to ameliorate the adverse effect of theforeign antibody sufficiently to permit multiple treatments with thetargeting agent.

None of these approaches has proven altogether satisfactory. Animportant need persists for a means to reduce or eliminate the adverseimmune response to targeting antibody and antibody conjugates in orderto gain the full benefit of these diagnostic and therapeutic agents.

This goal has been achieved with the CDR-grafted humanized murineanti-human CEA mAbs that are described below.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a humanized ClassIII anti-CEA mAb in which the CDRs of a murine Class III anti-CEA mAb(MN14) are functionally engrafted to the amino acid sequence of a humanantibody or antibody fragment to provide an immunological reagent withthe anti-CEA binding properties of the murine Class III, anti-CEA mAband the immunogenic properties of a human mAb in a human patient.

It is another object of the present invention to provide DNA constructsencoding such antibodies. Particular objects in this regard aresubstrate DNAs that facilitate genetic manipulation to produce improvedantibodies and DNAs encoding the antibodies with advantageous propertiesin cell culture and antibody production.

Yet another object of the invention is to provide vectors forpropagating the DNA and for expressing the antibody. A related object ofthe invention is to provide cells containing a vector for the purposesof storage, propagation, antibody production and therapeuticapplications.

Still another object of the invention is to provide compositionscomprising the antibodies for use in diagnosis and therapy. In thisregard it is an object of the invention to provide conjugates comprisingthe antibodies complexed with imaging agents and therapeutic agents forex vivo and in vivo imaging, diagnosis, prognosis and therapy, amongothers.

In accomplishing the foregoing objects, there has been provided, inaccordance with one aspect of the present invention, a humanized mousemAb, comprising the CDRs of a murine Class III, anti-CEA mAb (MN-14)engrafted to the framework regions of a heterologous (human) antibody,wherein the thus humanized mAb antibody retains the Class III, anti-CEAbinding specificity of the murine mAb but in the patient is lessimmunogenic than is the parent MN-14 murine monoclonal antibody.

In a highly preferred embodiment, the light chain variable regions ofthe humanized antibody are characterized by the formula:FR_(L1)-CDR_(L1)-FR_(L2)-CDR_(L2)-FR_(L3)-CDR_(L3)-FR_(L4)wherein each FR is separately a framework region of a human antibody,and each CDR is separately in a complementarity-determining region ofthe light chains of MN-14, and the subscripts refer to light (“L”) chainregions. The heavy chain variable regions are characterized by theformula:FR_(H1)-CDR_(H1)-FR_(H2)-CDR_(H2)-FR_(H3)-CDR_(H3)-FR_(H4)wherein FR and CDR have the same meanings as above, and wherein thesubscripts “H” refer to heavy chain regions.

In one embodiment, CDR_(L1) has the amino acid sequence KASQDVGTSVA(SEQ. ID NO. 20); CDR_(L2) has the amino acid sequence WTSTRHT (SEQ. IDNO. 21); CDR_(L3) has the amino acid sequence QQYSLYRS (SEQ. ID NO. 22);CDR_(H1) has the amino acid sequence TYWMS (SEQ. ID. NO. 23); CDR_(H2)has the amino acid sequence EIHPD SSTINYAPSLKD (SEQ. ID NO. 24); and,CDR_(H3) has the amino acid sequence LYFGFPWFAY (SEQ. ID NO. 25).

In another embodiment, FR_(L1) has the amino acid sequence DIQLTQSPSSLSASVGDRVTITC (SEQ. ID NO. 26); FR_(L2) has the amino acid sequenceWYQQKPGKAP KLLIY (SEQ. ID NO. 27); FR_(L3) has the amino acid sequenceGVP(S or D)RFSGS(G or V) SGTDFTFTISSLQPEDIATYYC (SEQ. ID NO. 28);FR_(L4) has the amino acid sequence FGQGTKVEIK (SEQ. ID NO. 29); FR_(H1)has the amino acid sequence EVQLVESGGG VVQPGRSLRLSCSSSGFDFT (SEQ. ID NO.30), EVQLVESGGGVVQPGRSLRLSCSAS GFDFT (SEQ. ID NO. 31), or QVQLQ ESGPGLVRPS QTLSL TCTSS GFDFT (SEQ. ID NO. 32); FR_(H2) has the amino acidsequence WVRQAPGKGLEWVA (SEQ. ID NO. 33), WVRQAPGKGLEWIA (SEQ. ID NO.34), or WVRQPPGRGLEWIA (SEQ. ID NO. 35); FR_(H3) has the amino acidsequence RFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ. ID NO. 36),RFTISRDNAKNTLFLQMDSLRPEDTGVYFC AS (SEQ. ID NO. 37), orRVTMLRDTSKNGSFLRLSSVTAADTAVYYCAS (SEQ. ID NO. 38); and FR_(H4) has theamino acid sequence WGQGTPVTVSS (SEQ. ID NO. 39), or WGQGTTVTVSS (SEQ.ID NO. 40); and wherein C may be in the sulfhydryl or disulfide form.

Another preferred embodiment comprises a diagnostic or therapeutic agentcomplexed to Class III, anti-CEA humanized mAb in which the CDRs of theantibody are derived from those of the MN-14 murine mAb and the FRs arederived from those of the heterologous (human) antibody, wherein theconjugate retains the Class III, anti-CEA binding specificity of MN-14,but is in humans less immunogenic than is murine MN-14. In one suchembodiment the light chain and heavy chain variable regions arecharacterized as shown above and have amino acid sequences also asdescribed above.

In yet another preferred embodiment, a method for diagnosing or treatinga patient comprises the step of administering in an appropriate regimenthe conjugate of the previous preferred embodiment.

Another preferred embodiment comprises an isolated, purified DNA thatencodes the light chain, the heavy chain or both chains of the humanizedantibody described above.

Another preferred embodiment comprises the DNA sequence of the CDRs andFRs described above.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description and appendedclaims.

Illustrative Glossary

The following terms or abbreviations are used in the presentapplication. The meanings set out in this glossary are for illustrativepurposes only. The full meaning of the terms will be apparent to thoseof skill in the art.

“CDR” is used as an abbreviation for Complementarity Determining Region.These are the regions within the variable regions of an antibody thatare primarily, but not exclusively, responsible for antigen-antibodybinding.

“FR” is an abbreviation for Framework Region. Broadly speaking, theseare the portions of the variable regions of an antibody which lieadjacent to or flank the CDRs. In general, these regions have more of astructural function that affects the conformation of the variable regionand are less directly responsible for the specific binding of antigen toantibody, although, nonetheless, the framework regions can affect theinteraction.

“Chimeric” refers to an antibody in which the variable region is derivedfrom a mouse antibody and the constant region is derived from anantibody from a heterologous (other) species.

“Humanized” refers to a chimeric antibody as defined above, but one inwhich the FR variable regions are derived from a human antibody.

“HAMA” refers to human antibodies directed to a mouse antibody, that areproduced when a mouse antibody is administered to a human subject.

“HAHA” refers to human antibodies directed to a humanized mouseantibody.

“CEA” refers to carcinoembryonic antigen, a 180 kDa glycoprotein that isexpressed in most adenocarcinomas of endodermally-derived digestivesystem epithelia and in some other cancers such as breast cancer andnon-small cell lung cancer.

The letter “h” as a prefix means “humanized”.

Other abbreviations are used in accordance with Roitt et al, IMMUNOLOGY,3rd ed. Mosby Year Book Europe Ltd. (1993), the entirety of which isherein incorporated by reference.

These and other terms used in the present disclosure are used in thesame sense as ordinarily they are employed in the arts to which thisinvention pertains.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B (SEQ. ID. NOS. 1 and 2) shows the consensus DNA sequenceof murine NEWM MN-14 variable region heavy chain (“VH”) and its proteintranslation product. The CDRs are enclosed in boxes.

FIGS. 2A and 2B (SEQ. ID. NOS. 3 and 4) shows the consensus DNA sequenceof murine MN-14 variable region light chain (“VK”) and its proteintranslation product. The CDRs are enclosed in boxes.

FIG. 3 shows a vector for the expression of chimeric or humanized MN-14heavy chain gene. The schematic diagram shows both the chimeric and areshaped heavy chain immunoglobulin (Ig) gene and the pSVgpt expressionvector. The diagram at top, labeled “CHIMERIC,” is a map showing DNAencoding the MN-14 mouse VH region joined to DNA encoding a human IgG1constant region. In the MN-14 VH region the three CDRs are indicated bythe three dark areas. The FRs are indicated by the four stippled areas.The middle diagram, labeled “RESHAPED” shows the humanization of theMN-14 VH region in which the mouse FRs have been replaced by human FRs,indicated by the four clear areas in the “Human” VH region. The circularmap of the expression vector pSVgpt at bottom shows the HindIII/BamHIinsertion site for the reshaped MN-14 antibody gene just downstream froman Igh enhancer element. The map also indicates some importantfunctional domains in the vector, including the replication origins forpropagation in E. coli (colEl ori) and in mammalian cells (SV40 promoterregion), and genes encoding selective markers for culturing bacterial(Ap^(r)) and mammalian (gpt) cells transformed with the vector.Expression of the antibody gene in this case is mediated by the Igpromoter indicated by the solid circles near the HindIII site in themaps of the antibody genes.

FIG. 4 shows a vector for the expression of chimeric or humanized MN-14kappa chain gene. The diagram shows a chimeric and a reshaped MN-14kappa light chain gene and the pSVhyg expression vector. The diagram attop, labelled “CHIMERIC,” is a map showing DNA encoding the MN-14 mousekappa light chain variable region (“VK”) joined to DNA encoding a humankappa constant (“CK”) region. In the mouse VK region the three CDRs areindicated by the three dark areas and the FRs are indicated by the fourstippled areas. The middle diagram, labelled “RESHAPED” shows thehumanization of the MN-14 VK region in which the mouse FRs have beenreplaced by human FRs, which are indicated by the four clear areas inthe “Human” VK region. The circular map of the expression vector pSVhygat bottom shows the HindIII/BamH1 insertion site for the reshaped MN-14antibody gene just downstream from an Igh enhancer element. The mapindicates some important functional domains in the vector, including thereplication origins for E. coli (col E1 ori) and mammalian propagation(SV40 promoter region), and genes encoding selective markers forculturing bacterial (Ap^(r)) and mammalian (gpt) cells transformed withthe vector. Expression of the antibody gene in this case is mediated bythe Ig promoter schematized by the solid circles near the HindIII sitein the maps of the antibody genes.

FIGS. 5A and 5B shows the alignments of the murine MN-14 variableregions (SEQ. ID. NOS. 2 and 4) with the human variable regions NEWM VH(SEQ. ID NO. 5) and REI VK (SEQ. ID NO. 6) (FIG. 5A). and with the humanKOL VH region (SEQ. ID NO. 7) (FIG. 5B). CDRs are boxed, and the murineVH FRs, which are incorporated into the humanized VH, are marked withtheir positions according to the numbering system of Kabat et al.SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. GovernmentPrinting Office, Washington, D.C., 1987. Murine residues outside theCDRs that were included in the KLHuVH are indicated by a filled circle.

FIGS. 6A, 6B and 6C shows a comparison of the amino acid sequencebetween murine (SEQ. ID NO. 2) and humanized (SEQ. ID NOS. 57, 8-11, 58and 12-15). MN-14 VH framework residues (FR). Only human FR residuesdifferent from the mouse are shown. CDAs for NEWM and KOL are also notshown. The position of the substitution is indicated according to theKabat et al. numbering system. The 3 CDRs are boxed.

FIG. 7 shows the DNA sequence and corresponding amino acid sequence(SEQ. ID NOS. 16 and 17) of the MN-14HuVH (KLHuVh) region. CDRs areboxed.

FIG. 8 shows the DNA sequence and corresponding amino acid sequence(SEQ. ID NOS. 18 and 19) of the MN-14HuVK region. CDRs are boxed.

FIG. 9 is a graph of MN-14 blocking (i.e., competition) assays comparingrelative binding affinities of KLHuVH variants, including: (-∘-)KLHuVH/HuVK; (-▪-) KLHuVHAIG/HuVK; (-●-) KLHuVHAIGAY/HuVK; (-□-)KLHuVHAIGA/HuVK; (-▴-) chimeric control; and, (-Δ-) murine control.

FIG. 10 shows MN-14 blocking assays comparing hMN-14 with HMN-14-NVT(glycosylated in FR 1 region).

FIG. 11 is a radioautogram of the abdomen of a colon cancer patientfollowing administration of ¹³¹I-labelled hMN14IgG (left panel) ormMN14IgG (right panel) in the same patient.

DETAILED DESCRIPTION OF THE INVENTION

Notwithstanding past failures to develop an effective non-HAMA-inducinganti-CEA antibody having the CEA-binding characteristics of MN-14, ithas been discovered that the CDRs of the MN-14 mAb can be grafted ontothe FRs of a human antibody to provide antibodies and antibody-derivedreagents that have the antigen binding properties of the MN-14 anti-CEAmAb, while also exhibiting reduced induction of HAMA and augmentedeffector activities.

The murine anti-CEA IgG1 monoclonal antibody MN-14, and its production,have been described previously. Hansen et al., Cancer, 71: 3478 (1993);Primus et al., U.S. Pat. No. 4,818,709. MN-14 meets all of the criteriaof a Class III, anti-CEA monoclonal antibody, being unreactive withmeconium antigen by EIA and not reacting with normal tissues.

Blocking studies are carried out according to Hansen et al. 1993, above,Losman et al., Int. Cancer, 56: 580 (1994); Hansen et al., Clin. Chem.,35: 146 (1989). Using the same conditions as described in thosereferences for quantification of CEA, binding of humanized MN-14 may beassessed relative to a labeled MN-14 probe. A typical probe is MN-14conjugated to horse radish peroxidase (HRP). Both labeled and unlabeledMN-14 are added to a CEA sample fixed to a solid support such asmicrotitre plate wells. The degree of “blocking” of labeled MN-14binding to CEA is a direct reflection of unlabeled MN-14 activity. Usingstandard MN-14, the relative activity of an unknown sample of humanizedMN-14 or derivatives thereof can be determined. Typically, the reactionsare performed in the wells of a microtitre plate where wells are chargeddirectly with CEA at a level of, for example, 25 μg/well or indirectlywhere the wells are precharged with an antibody reactive with CEA but toan epitope different than that to which MN-14 interacts; such anantibody may be the MN-15 mAb. CEA can thus be indirectly fixed to thewell. A competitive binding EIA assay can then be performed with such acharged plate.

Alternate to the aforementioned HRP-labeled mAbs, antibodies can beradioiodinated conventionally with, for example, ¹³¹I by thechloramine-T method to a specific activity of about 10 mCi/μg, and freeradioisotopes removed by chromatography on acrylamide gel columns (seeHansen et al., 1993, above).

Molecular biological techniques suitable to carrying out the inventionas herein described also are known to those skilled in the art. Suitableteachings are described in numerous manuals and primary publications,including inter alia, Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al.,Eds., Green Publishing Associates and Wiley-Interscience, John Wiley andSons, New York (1987, 1988, 1989), which are herein incorporated byreference in their entirety including supplements.

MN-14 light and heavy chain CDRs disclosed herein, and modified MN-14CDRs can be integrated into other antibodies using well-knownrecombinant techniques, such as those described in the above references.

Specific methods suitable to this end are shown below in the examples.Based on the amino acid sequences set forth herein, oligonucleotidesencoding MN-14 CDRs can be synthesized. Oligonucleotides that encodemodified CDRs may be made, as well as those that encode exactly theamino acid sequences herein set forth. Also, the oligonucleotides maycontain nucleotides in addition to those of an MN-14 CDRs, to facilitatecloning, for instance. Oligonucleotide synthesis techniques are wellknown, and can be carried out on automated equipment available from anumber of manufacturers. Moreover, oligonucleotides of any specifiedsequence can be obtained commercially.

Oligonucleotides encoding the MN-14 CDRs and/or specific FR residues. orrepresenting the complementary strand thereof, may be used to introducecodons for these residues into VH or VK DNA by site-directed mutagenesisprovided that the ends of the oligonucleotides, generally 12nucleotides, are designed to anneal perfectly to the template DNA. Thetemplate DNAs are typically single-stranded DNAs representing M13vectors that carry a variable region DNA encoding the required FRs. Inone method, the mutagenic oligonucleotides are phosphorylated at their5′ ends and, together with an oligonucleotide priming 5′ to variableregion DNA, are annealedd to the ssDNA template. The oligonucleotidesare extended using T7 polymerase and the fragments linked together by T4DNA ligase to give a complete mutant strand covering the whole variableregion. Using the mutant strand as a template, multiple copies of itscomplementary strand can be synthesized from a suitable primer using TaqDNA polymerase in a thermal cycling reaction. Once the mutant strand hasbeen preferentially amplified in this manner, the DNA can be amplifiedby conventional PCR for cloning, sequencing and expression.

Suitable antibody-encoding DNAs are illustrated by the disclosureherein, but include practically any such DNA. A variety of humanantibody genes are available in the form of publically accessibledeposits. Many sequences of antibodies and antibody-encoding genes havebeen published and suitable antibody genes can be synthesized from thesesequences much as described above.

The scope of this invention encompasses all alleles, variants andmutations of the DNA sequences described herein.

CDR grafting in accordance with the present disclosure may be carriedout using established techniques. Antibody-producing cell lines may beselected and cultured using techniques well known to the skilledartisan. Such techniques are described in a variety of laboratorymanuals and primary publications. For instance, techniques suitable foruse in the invention as described below are described in CURRENTPROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds., Green PublishingAssociates and Wiley-Interscience, John Wiley and Sons, New York (1991)which is herein incorporated by reference in its entirety, includingsupplements.

RNA may be isolated from the original hybridoma cells by standardtechniques, such as guanidinium isothiocyanate extraction andprecipitation followed by centrifugation or chromatography. Wheredesirable, mRNA may be isolated from total RNA by standard techniquessuch as chromatography on oligodT cellulose. Techniques suitable tothese purposes are well known in the art as described in the foregoingreferences.

cDNAs that encode the light and the heavy chains of the antibody may bemade, either simultaneously or separately, using reverse transcriptaseand DNA polymerase in accordance with well known methods. It may beinitiated by consensus constant region primers or by more specificprimers based on the published heavy and light chain DNA and amino acidsequences.

PCR also may be used to isolate DNA clones encoding the antibody lightand heavy chains. In this case the libraries may be screened byconsensus primers or larger homologous probes, such as mouse constantregion probes. The necessary techniques are well known to those of skillin the art, are set forth in the foregoing Sambrook and Ausubelreferences and are illustrated by the examples set forth below.

cDNAs that encode the light and the heavy chain of an antibody can bepropagated in any suitable vector in any suitable host prior toisolation of the CDR. Often the clones will most conveniently bepropagated for this purpose in E. coli as illustrated in the examplesbelow. However, a variety of other vectors and host cells well knowN tothose of skill profitably may be employed in this aspect of theinvention. A variety of such vectors are described in the foregoingreferences.

DNA, typically plasmid DNA, may be isolated from the cells, restrictionmapped and sequenced in accordance with standard, well known techniquesset forth in detail in the foregoing references relating to recombinantDNA techniques.

DNAs encoding antibody heavy and light chains and fragments thereof inaccordance with the vector are used to construct chimeric andCDR-grafted humanized MN-14 antibodies.

The CDRs of the MN-14 anti-CEA mAb are herein identified and described,and illustrated in FIGS. 1A and 1B and and 2A and 2B (SEQ. ID NOS. 2 and4, respectively) Using these sequences, CDRs of the MN-14 heavy andlight chain can be synthesized for use in the present invention. It isnot necessary to redone MN-14 CDRs from a natural source. The DNA andamino acid sequences are set forth herein. Oligonucleotide synthesistechniques suitable to this aspect of the invention are well known tothe skilled artisan and may be carried out using any of severalcommercially available automated synthesizers. In addition, DNAsencoding the CDRs set forth herein can be obtained through the servicesof commercial DNA synthesis vendors.

Polynucleotides synthesized in accordance with this aspect of theinvention may include those not derived from an MN-14 CDR as well asthose that make up the CDR. The additional bases may be included tofacilitate joining the CDR to the FRs from a heterologous source. Theymay comprise restriction sites or overlapping complementary regions forthis purpose. The synthesis of longer, double-stranded DNAs fromshorter, overlapping, single-stranded DNAs is well known to those ofskill in the art. Likewise, well known is the end-to-end joining ofDNAs, including blunt-ended DNAs and those with at least partiallyoverlapping complementary termini. These techniques are illustrated inthe foregoing references on recombinant DNA techniques, for instance.

The CDRs of the MN-14 heavy and light chains may also be modifiedparticularly after incorporation into a chimeric or humanized antibodyusing well-known recombinant DNA techniques for deleting, inserting andaltering bases in a cloned or synthetic DNA or RNA. Site-specificmutagenesis techniques suitable to this end are well known to those ofskill in the art, and are illustrated in the foregoing references onrecombinant DNA techniques. Also illustrated are deletional andinsertional techniques. These methods can be used to introducepractically any desired alteration into polynucleotides that encode theMN-14 CDRs or into other regions of a closed heavy or light chain gene.

MN-14 CDRs and modified MN-14 CDRs can be introduced into practicallyany set of FRs in accordance with the present invention. It will beappreciated by those of skill in the art that a variety of well knowntechniques for cloning and manipulating polynucleotides may beeffectively employed in this regard. Such techniques are illustrated bythe methods set forth in the foregoing recombinant DNA-relatedreferences.

In a particularly preferred embodiment of the present invention, MN-14CDRs are grafted into a human antibody. It will be understood that humanantibody in this context refers to any antibody that occurs in a humanor an engineered antibody that has been designed, in some respect, to becompatible with the human immune system. Particularly preferred for thispurpose are antibodies that, broadly, do not engender an adverse immuneresponse in a patient. More particularly, the expression “humanantibody” is intended to mean an antibody encoded by a gene actuallyoccurring in a human, or an allele, variant or mutant thereof.

Once DNA encoding an MN-14-derived CDR-grafted antibody has beenassembled from MN-14 VH and VK region DNAs and the variable regions thusformed combined with their respective light and heavy chains of humanconstant domains, it may be inserted into a vector for propagation andexpression by conventional techniques. In this manner desired amounts ofthe antibody may be obtained.

The MN-14 CDR-grafted human antibody can be used in imaging applicationsby administrating to a subject the humanized antibody or Fab′ thereofconjugated with an imaging compound or isotope.

The antibody is conjugated to a label for imaging using conventionalmethods. Such conventional methods include, but are not restrictedto: 1) direct radioiodination of the antibody protein or fragmentsthereof or 2) direct attachment to the antibody or fragments thereof ofmetallic nuclides (see, e.g., Hansen et al., Cancer, 73: 761 (1994)).The use of bifunctional chelates that can be used to bind variousdiagnostic or therepeutic metals to the antibody or fragment thereof isalso within the scope of the present invention (see, Antibodies inRadiodiagnosis and Therapy, ed. M. R. Zalutsky, 1989, CRC Press, BocaRaton, Fla., and Cancer Therapy with Radiolabeled Antibodies, ed. D. M.Goldenberg, 1994, CRC Press, Boca Raton, Fla.). Following theconjugation procedure and characterization of the product,satisfactorily labelled conjugates are purified to homogeneity underconditions that conform to Good Manufacturing Procedures (“GMP”)appropriate to the production of diagnostic compositions for use inhuman patients.

The reaction of serum antibody with the MN-14 CDR-grafted antibody andimaging agent portions of the conjugate can be determined over thecourse of the diagnostic procedures, including the reaction of controlsera obtained prior to administration of conjugate. Similardeterminations are made in other patients treated with similarconjugates of MN-14 itself. The sera antibody reactive with CDR-graftedMN-14 human antibodies detected by these tests is much less than theantibody reactive with antibody portion of the conjugate in patientstreated with the murine MN-14-containing conjugates.

Humanized MN-14 antibodies conjugated to aminodextran and to boron maybe used for diagnostic purposes. MN-14 and CDR-grafted MN-14 antibodiescan be prepared as set forth above for conjugation to anaminodextran-boron adduct. Amino-dextran-boron adducts can be preparedby reaction of a suitable boron cage compound (e.g., a 12-boroncarborane suitably derivatized with an amino-dectran functional group).In a preferred embodiment, the amino-dextran is reacted with an excessof a haloacetyl acid ester or anhydride (such as iodoacetic anhydride),thereby producing an amino-dextran with a desired number of haloacetylgroups, usually ranging from 10-1000 groups, depending on the reactionconditions and the size of the amino-dextran. A suitable boronderivative such as mercaptocarborane-B12 is reacted, in a desired molarexcess, with the haloacetyl-amino-dextran via an alkylation reaction. Ina preferred embodiment, a number of haloacetyl groups on the boronatedhaloacetyl amino-dextran remain unreacted, and can be used as a “handle”to attach the adduct to protein thiol groups.

MN-14 CDR-grafted humanized antibodies and their derivatives, because oftheir reduced immunogenicity, are useful in therapy, for passiveimmunization without negative immune reactions such as serum sickness oranaphylactic shock, for localization and in vivo imaging of tumors asdescribed above, for specific treatment of disease cells, e.g., sitedirected delivery of cytotoxins, immunomodulators or otherpharmaceutically active molecules where local concentration of theactive agent is an important factor, or the like, thereby establishingthe practical utility of these humanized antibodies. As described above,for in vivo imaging, the humanized, CDR-grafted MN-14 monoclonalantibody is radiolabeled or conjugated with a metal chelator complexedwith a radionuclide, e.g., iodine, ytrium, technetium, or the like, andradio-scanning techniques may be used to detect primary and metastaticCEA tumors. To that end, the radioactive antibody is injected, e.g.,intravenously, and the patient scanned with a gamma imager at regularintervals. Tumors expressing CEA will take up more radioantibodies thanother tissues and will be easily recognized by the imaging camera.Preferentially, monoclonal antibodies labelled with ¹³¹I are used inamounts of 3 to 10 μg representing 15 to 30 μCi per kg body weight. Fortherapy with cytotoxic agents, the antibodies are conjugated to any of avariety of known therapeutic agents such as doxorubicin, methotrexate,taxol, ricin A, radioactive atoms, cytoxic agents, and the like,formulating such conjugate in a pharmaceutically acceptable sterilevehicle, and administering the formulation by conventional means. Thetherapeutic dosages can be readily determined conventionally by the userof average skills in these arts. The therapeutic dose for mammals isbetween about 1 mg and 5 mg per kg body weight for the monoclonalantibodies themselves, and between 0.1 mg and 5 mg per kg body weightfor conjugates with cytotoxic drugs, depending on the status of thepatient and the mode of administration. Alternately, the humanizedantibodies can be used in combination with components of the host'simmune system, e.g., complement or cell mediated responses, in order toremove from the subject CEA-presenting cancer cells. The immuneresponses of patients may be monitored in accordance with the foregoingprocedures. For additional procedures for radioimaging and therapy, seeEP 0 323,806, Hansen et al., Cancer 71: 3478-85 (1993), and U.S. Pat.No. 4,818,709 and references contained therein, all of which areincorporated by reference.

Preferred are pharmaceutical preparations for parenteral administration,such as are described in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., 1989. The final preparations contain from0.01% to 50% of active ingredients. Methods for the production of suchconjugates and their use in diagnostics and therapeutics are providedin, for example, Shih et al., U.S. Pat. No. 5,057,313; Shih et al., Int.J. Cancer 41:832 (1988); copending, commonly owned U.S. Ser. No.08/162,912, now U.S. Pat. No. 5,443,953; and, McKearn et al., U.S. Pat.No. 5,156,840, the contents of which are incorporated by reference.

As noted above, for purposes of therapy, a humanized antibody conjugateand a pharmaceutically acceptable carrier are administered to a patientin a therapeutically effective amount. A combination of a conjugate anda pharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is “physiologically significant”if its presence results in a detectable change in the physiology of arecipient patient. A targeted therapeutic agent is “therapeuticallyeffective” if it delivers a higher proportion of the administered doseto the intended target than accretes at the target upon systemicadministration of the equivalent untargeted agent.

To be therapeutically effective the conjugate and carrier may need to beadministered in combination with other therapeutic agents or as part ofa broader treatment regimen. Physicians are currently of the opinionthat the effectiveness of targeted therapeutics can often be greatlyincreased when used in a combination therapy approach. For example,high-dose radioimmunotherapy for B-cell lymphomas, which causes severehematologic toxicity when used alone, has been shown to be highlyeffective when used in combination with autologous bone marrowreinfusion. Press et al., “Treatment of Relapsed B Cell Lymphomas withHigh Dose Radioimmunotherapy and Bone Marrow Transplantation” in CANCERTHERAPY WITH RADIOLABELED ANTIBODIES, Goldenberg, ed. (CRC Press, BocaRaton, 1994) ch. 17. In another example a five-fold enhancement of tumoruptake of a radiolabeled antibody is observed when the tumor ispreirradiated. Leichner et al., Int. J. Radiat. Oncol. Biol. Phys.14:1033 (1987). Mechanisms which have been shown to have the potentialfor improving the clinical efficacy of radioimmunotherapy are alsodiscussed in DeNardo et al., “Overview of Obstacles and Opportunitiesfor Radioimmunotherapy of Cancer” in CANCER THERAPY WITH RADIOLABELEDANTIBODIES, Goldenberg, Ed. (CRC Press, Boca Raton, 1994) ch. 11.Methods of developing such combination protocols, as well as toinvestigate dose-limiting side effects and to potentiate and amplifytargeting, uptake, and beneficial side effects, are well known toskilled clinical artisans in this field and would not require undueexperimentation to develop.

In vivo experiments using conjugates of the humanized MN-14 withdiagnostic and therapeutic agents have been carried out with animalmodels and with human patients (see Example 11 below). The CDR-graftedhumanized antibody conjugate exhibited a better therapeutic profile andcould be used in longer treatment regimens than the parental MN-14antibody conjugate. The CDR-grafted antibody conjugate provided a bettertherapeutic effect and fewer deleterious side effects than the controlmurine antibody conjugates.

For example, the antibody was covalently complexed toaminodextran-conjugated methotrexate using the method described by Shihet al., above using carbohydrate hydroxyl groups for derivatizationpurposes. In order to determine the contribution of antibodycarbohydrate groups on immunoactivity, a mutation can be introduced atposition 18-20 in the VK FR1 region of hMN-14 (the prefix “h” isintended to mean “humanized”) so as to introduce a glycosylation site,NVT, prior to expression of the blocking gene in mammalian cells.Comparison of hMN-14 antibody with mutated hMN-14-NVT in a blocking cellbinding assay (FIG. 8) has demonstrated that the carbohydrate moiety atposition 18 is without influence on immunoreactivity of this humanizedantibody.

Aminodextran, average molecular mass 40 kDa, is oxidized by NaIO₄ toform aldehydes (by the oxidation of hydroxyl groups). About 50 to 150moles of aldehydic groups are introduced per mole of aminodextran bycareful control of the reaction conditions and timing. The aldehydesthen are reacted with an excess of 1,3-diamino-2-hydroxypropane to formSchiff bases with virtually all of the aldehydes. The Schiff bases arethen reduced by treatment with excess NaBH₄. The amine-derivatizeddextran then is purified by gel-exclusion chromatography.

The cytotoxic drug methotrexate (MTX) is activated by treatment withdicyclocarbodiamide, followed by reaction with N-hydroxysuccinimide,both in dimethylformamide. Activated MTX is mixed in a 50:1 ratio withthe amino derivatized dextran in aqueous solution. The product provides,after purification, MTX-derivatized dextran having about 35 MTX molesper mole. The MTX adduct thus obtained is conjugated to a MN-14CDR-grafted antibody using methods described in Shih, et al., supra. Forexample, the antibody carbohydrates are oxidized and the resultantaldehydes are reacted with the remaining amines on the dextran in theadduct. The Schiff-base product obtained thereby is reduced by treatmentwith sodium cyanoborohydride in 10-fold molar excess over antibody. Thereduced antibody-dextran-MTX product is thoroughly purified prior toassay, and formulated for administration to patients.

Parental MN-14 antibody is conjugated to dextran-MTX in the same way, asa control.

The purified CDR-grafted antibody conjugate can be administered topatients with a CEA-producing cancer (see above). The response totherapy is monitored, including adverse side effects, particularly thosewhich are mediated by the patient's immune systems. Patients treatedwith the CDR-grafted antibody conjugate show improved therapeuticresults, decreased immune response to the agent and notably decreasedimmune-mediated adverse effects of therapy. Therapy with the CDR-graftedantibody conjugate can be carried out at higher dosages and for longerperiods of time then with the parental murine MN-14 antibody, allowingmore aggressive therapies and improved responses.

The present invention is further described by reference to thefollowing, illustrative examples. It will be appreciated that thetechniques related to isolating DNA clones encoding MN-14 light andheavy chain genes are illustrated by cloning techniques useful toisolate light and heavy chain genes of any antibody from producingcells. There is no necessity, given the disclosed sequence, to reisolateMN-14 heavy and light chain genes to carry out the invention.

It should be understood that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from the following illustrativedescription.

EXAMPLE 1 Culturing Antibody Producer Cells

A mouse/mouse hybridoma cell line producing Class III, anti-CEAmonoclonal antibodies was established according to Hansen et al. (1993)above and Primus et al. (1983) above.

Cells were selected for secretion of kappa IgG1 by testing conditionedmedium using standard isotyping techniques. A variety of kits for thispurpose are commercially available. Such cells were screened forproduction of antibody by testing conditioned medium using a standardblocking assay described above. Stocks of producer cells that proved outin the assay were expanded and frozen in liquid nitrogen.

EXAMPLE 2 Isolating RNA from Producing Cell Lines

MN-14-producing cells were expanded in culture, collected bycentrifugation and washed. Total RNA was isolated from the cells in thepellet according to Favaloro et al., Methods in Enzymology 65: 718(1980) and Orlandi et al., Proc. Nat'l Acad. Sci., USA 86: 3833 (1989),which are incorporated by reference.

EXAMPLE 3 cDNA Synthesis and Amplification of the Heavy Chain VariableRegion

mRNA from MN-14 producing cells was used to synthesize cDNA usingstandard techniques of cDNA synthesis and DNA amplification by PCR, asdescribed below. In general, the primers used for PCR included arestriction endonuclease cleavage site at their 5′ ends to facilitatecloning of the amplification product. An oligonucleotide complementaryto the end of the sense strand of the DNA encoding the first constantregion domain of the murine IgG₁ heavy chain (“CH1”) was used to primefirst strand cDNA synthesis by reverse transcriptase. The sequence ofthis primer, CG1FOR, is shown in Table 1. Table 1 below provides otheroligonucleotide sequences used herein. TABLE 1 OLIGONUCLEOTIDE SEQUENCESSEQ. ID NO. CG1FOR 41 5′ GGAAGCTTAGACAGATGGGGGTGTCGTTTTG 3′ VH1FOR 42 5′TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 3′ VH1BACK 43 5′AGGTSMARCTGCAGSAGTCWGG 3′ SH1BACK 44 5′TGGAATTCATGGRATGGAGCTGGRTCWTBHTCTT 3′ SH2BACK 45 5′TGGAATTCATGRACTTCDGGYTCAACTKRRTTT CK2FOR 46 5′GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′ VK1FOR 47 5′ GTTAGATCTCCAGCTTGGTCCC3′ VK3FOR 48 5′ GTTAGATCTCCAGTTTGGTGCCT 3′ VK1BACK 49 5′GACATTCAGCTGACCCAGTCTCCA 3′ VK2BACK 50 5′ GACRTTCAGCTGACCCAGGMTGMA 3′VK3BACK 51 5′ GACATTCAGCTGACCCA 3′ VK4BACK 52 5′GACATTGAGCTCACCCAGTCTCCA 3′ VK5BACK 53 5′TTGAATTCGGTGCCAGAKCWSAHATYGTKATG 3′ VK6BACK 54 5′TTGAATTCGGTGCCAGAKCWSAHATYGTKCTC 3′ VK7BACK 55 5′TTGAATTCGGAGCTGATGGGAACATTGTAATG 3′ VK8BACK 56 5′CWGAGAAATTCAGCTGACCCAGTCTC 3′

Restriction sites incorporated in primers to facilitate cloning areunderlined.

The variable region of the heavy chain (“VH”) cDNA then was amplified bythe PCR using the same primer, CG1FOR, and a primer based on theconsensus sequence of the 5′ end of VH genes (VH1BACK), as described inOrlandi et al. (1989) cited above. The PCR product of this reaction wasanalyzed by agarose gel electrophoresis, which, upon ethidium bromidestaining and fluorescence illumination, revealed one major band of about400 bp, as expected.

For confirmatory sequences from a second cDNA preparation, signalsequence primers were used in the PCR to allow determination of theauthentic amino acids of the N-terminus. SH1BACK and SH2BACK, degenerateoligonucleotides based on heavy chain signal sequence coding regions,were used in separate reactions in concert with CG1FOR. A diffuseproduct band was obtained from CG1FOR, SH1BACK amplification.

In order to increase the VH content of the product it was excised fromlow melting point agarose and amplified using SH1BACK and anoligonucleotide complementary to a fourth framework region consensussequence, VH1FOR. This product of this reaction was a discrete band whenanalyzed by agarose gel electrophoresis.

EXAMPLE 4 Cloning and Sequencing DNA Encoding the MN-14 Heavy ChainVariable Region Obtained by PCR

The amplification product obtained using the CG1FOR, VH1BACK primer pairwas digested with HindIII and PstI separately. The cleavage sites ofthese enzymes are included in the PCR primers. It was preferable todetermine whether there were also sites internal to the VH. Agarose gelanalysis of the restriction fragments indicated the presence of aninternal PstI site close to one end of the DNA. The PCR product wasdigested with HindIII and PstI, cloned into M13mp18 and 19 and the DNAsequence of the inserts of representative clones determined. Themajority of the clones contained inserts of the same VH DNA.

The sequencing confirmed the presence of this additional, unexpectedPstI site, which was close to the 3′ end of the sequence of the CG1FORprimer partially encoding the final two amino acids of the VH. Althoughseveral full length VH clones were obtained by this method, further PCRproduct DNA was cloned as PstI-PstI fragments. These clones thuscontained full VH sequences but none of the constant region given byCG1FOR. A total of 16 full-length clones were obtained from the VK1BACK,CG1FOR product. In these experiments about 25% of the clones that wereanalyzed contained inserts unrelated to the VH region.

In order to confirm the VH sequence from a second cDNA preparation and,at the same time, to obtain the authentic, rather than primer-dictated,DNA sequence corresponding to the N-terminus of the VH, the PCR productfrom VH1FOR and SH1BACK primers was cloned. These primers contain BstEIIand EcoR1 restriction sites, rather than the PstI and HindIII sites ofCG1FOR and VH1BACK described above. The PCR product of this reaction wascloned by digesting with BstEII, filling in the BstEII ends, digestingwith EcoRI, and ligating the EcoRI and blunt ends to the vector, whichhad been digested with EcoRI and HindIII. The sequences of the clonedfragments were determined. The yield of VH fragments was relatively low,perhaps reflecting a lack of specificity in the PCR caused by degeneracyof SH1BACK. However, four of the 18 clones that were sequenced containedDNA encoding the MN-14 VH region as previously sequenced. The otherinserts did not derive from VH-encoding DNA.

In all, 20 full-length MN-14 VH clones were obtained. Five transitionmutations were observed amongst the sequences in the MN-14 VH regionclones. These mutations are likely to have been introduced duringamplification as a result of misincorporation by Taq polymerase.

EXAMPLE 5 Analysis of the Amino Acid Sequence of the Heavy ChainVariable Region of MN-14

The amino acid sequence of murine MN-14 heavy chain variable region,translated from the VH DNA sequence, is shown in FIGS. 1A and 1B (SEQ.ID. NOS. 1 and 2). Comparison of this sequence with sequencesrepresenting the murine VH subgroups indicated that the variable heavyregion of MN-14 belongs to subgroup IIIB (see, Kabat et al. SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Government Printing Office,1987).

The MN-14 CDR sequences are different from any of those reported byKabat et al. (1987), supra. Furthermore, the amino acids at fourpositions in the MN-14 heavy chain VH framework regions are differentfrom those in the framework regions of other subgroup IIIB VH sequences.These four substitutions (Ser 14, Thr 30, Ser 98 and Pro 108) have beenobserved in other murine VH regions outside the IIIB VH subgroup,however, with the proline in Kabat position 108 being the most unusual.

Any unusual residues in the VH or VK may represent somatic mutationswhich proved advantageous to the binding of murine MN-14.

EXAMPLE 6 cDNA Synthesis and Amplification of DNA Encoding the MN-14 VK

cDNA encoding the kappa light chain of MN-14 was cloned in much the samefashion as the cDNA encoding the variable region of the heavy chain, asdescribed above. Several primers were used to prime reversetranscriptase for synthesis of the first strand of the kappa chain cDNA.The sequence of one primer, CK2FOR, was derived from the sequence of the5′ end of the constant region of kappa light chain genes (“CK”). Thesequences of two other primers, VK1FOR and VK3FOR, were based on thesequence of the 3′ end of the variable region of kappa light chain genes(“VK”).

The first strand DNA product was amplified by PCR using a number ofprimer pairs. Synthesis in one direction was primed by the primers usedto make the first strand. Polymerization in the other, “backward,”direction initiated from a series of kappa light chain-specific primerswhich had sequences based on either the sequence at the 5′ end of the VKregion, VK1BACK, VK2BACK, VK3BACK, VK4BACK and VK8BACK, or the sequenceencoding the last four amino acids of the signal peptide and the firstfour amino acids of the variable region, VK5BACK, VK6BACK and VK7BACK.In addition, cDNA primed by CK2FOR also was amplified using VK1FOR andVK8BACK.

The amplification products were analyzed by gel electrophoresis in themanner described in the Examples above. The products from the reactionsprimed by VK1BACK, VK3BACK, VK5BACK, VK7BACK and VK8BACK gave rise tothe expected 350 bp band.

EXAMPLE 7 Cloning and Sequencing the MN-14 Kappa Light Chain VariableRegion Obtained by PCR

Selected PCR products were cloned into M13mp18 and 19 using therestriction sites included in the amplification primers in a mannersimilar to that described for VH in Example 4 above. Nucleotidesequencing revealed that most inserts were not VK-related. This is notuncommon when attempting to clone VK cDNAs and it appears to be moredifficult to design VK-specific primers than VH specific primers.

From the VK1FOR/VK8BACK combination, a VK cDNA insert was obtained, butthis did not yield a functional VK due to a frameshift within the cDNAencoding CDR_(L3) and absence of the invariant Cys at position 23. ThisVK cDNA has been isolated from other hybridoma cells and it is derivedfrom the Sp2/0 fusion partner. CK2FOR/VK1BACK product yielded a furtherfour different aberrant VK cDNA inserts, in this case lacking theconserved residues of framework 4. A fifth VK insert obtained using thisprimer pair was that of a functional VK with the exception of aframeshift at the 3′end of VK1BACK, a phenomenon apparently due tomismatch-induced slippage of the primer. This problem may be avoided bythe use of VK8BACK which does not extend as far into the VK gene.However, analysis of further clones from VK1FOR/VK8BACK product did notyield the desired insert.

In order to amplify preferentially the putative VK, VK3FOR was designedfrom its genuine fourth framework sequence, and synthesized as analternative to VK1FOR. This strategy proved successful whenamplification of VK3FOR-primed cDNA with VK3FOR and VK8BACK yielded 4clones containing the desired VK.

The DNA and amino acid sequence of the murine MN-14 kappa light chainvariable region is set out in FIGS. 2A and 2B (SEQUENCE ID. NOS. 3 and4). This MN-14 VK can be placed in Kabat's VK subgroup V. Only 3residues in the MN-14 VK framework regions (Met 10, Val 66 and Thr 76)do not appear in other members of this subgroup. Met 10 and Val 66 arethe most unusual of the three. They are not found in any murine VKlisted in Kabat. None of the MN-14 VK CDRs are previosuly-reportedsequences in Kabat.

EXAMPLE 8 Grafting of the MN-14 VH and VK CDRs into Variable Regions ofHuman Antibody 8.1. Construction of Chimeric Anitbody Expression Vectors

In the production of a chimeric antibody consisting of murine variableregions and human constant regions, testing alongside the parent mouseantibody served to check that the correct variable region cDNAs havebeen isolated. A successful chimeric antibody also acts as a usefulcontrol when assessing the binding of humanized versions. The schemeused in cloning the variable regions for expression is described byOrlandi et al. (1989) supra, and is illustrated in FIGS. 3 and 4.

VH DNA was amplified from the M13 clone MNVH41 using the PCR witholigonucleotides VH1BACK and VH1FOR. The PstI and BstEII restrictionsites in the primers allowed the VH to be inserted into M13VHPCR1 in thecorrect context for expression. At this point, the internal BamHIrestriction site of the VH was removed by site directed mutagenesis. Thereaction product, which encompassed the entire HindIII-BamHI fragment ofM13VHPCR1 was cloned into pSVgpt and the VH sequence confirmed.

The human IgG1 constant region gene, published by Takahashi et al.,Cell, 29: 671-679 (1962) above then was added to the construct as aBamHI fragment, which yielded the vector referred to aspSVgptMN14MuVHHuIgG1.

VK DNA was similarly obtained from the M13 clone MNVK154 by PCRamplification with the primers VK8BACK and VK3FOR and the PvuII,BglII-digested product cloned into M13VKPCR1, whereupon the sequence ofthe variable region was checked. The HindIII-BamHI fragment was excisedfrom RF DNA and transferred to the plasmid pSVhyg. The construct alreadycontains a human kappa constant region gene as described in Hieter etal., Cell, 22: 197-207 (1980). The final vector thus obtained wasdesignated pSVhygMN14MuVKHuCK.

8.2 Expression and Testing of the Hybrid Antibody

The HindIII-BamHI fragment of M13KLHuVHAIGA was inserted into a plasmidpSVgpt to yield the expression vector pSVgptKLHuAIGAHuIgG1. Similarly,the HindIII-BamHI fragment of M13HuVK was inserted into the plasmidpSVhyg to yield the expression vector pSVhygMN14REIHuVKHuCK. About 5 μgpSVgptMN14MuVHHuIgG1 and 10 μg pSVhygMN14MuVKHuCK DNAs were linearizedwith Pvul and transferred into about 10⁷ subconfluent SP2/0 myelomacells by electroporation conventionally using a BioRad Model 165BR1160Gene Pulser Electroporator with a single pulse of 170 V, 960° F. Cellswere selected for the expression of the gpt gene in 24-well plates byaddition of mycophenolic acid and xanthine to the DMEM+10% FCS growthmedium.

Wells which contained colonies of surviving cells were identified. Thesupernatant medium was removed from these wells and assayed for humanantibody. Colonies that secreted antibodies were expanded to give 0.5 Lof conditioned medium for isolation of larger amounts of antibody.

Antibody was purified conventionally from the medium by protein-Aagarose affinity chromatography, initially. The purified antibody wascharacterized further with reference to native MN-14 antibody, humanantibodies and other controls.

The antibody was also characterized by its reaction profile in a MN-14blocking assay, which provided an informative comparison of CEA bindingaffinities of the hybrid antibodies with CEA binding by the MN-14 murineantibody which served as a positive control.

8.3 Humanization of the MN-14 Antibody

The human NEWM VH, KOL VH and REI VK frameworks were chosen as the basisfor reshaping the antibody, as they are likely to be tolerated inhumans. Alignments of the MN-14 VH (SEQ. ID NO. 2) and VK (SEQ. ID NO.4) with these human variable regions are shown in FIGS. 5A and 5B (SEQ.ID. NOS. 5-7).

A. NEWM Based Humanization.

The starting points for the introduction of MN-14 CDRs are DNAs encodingthe required FRs and irrelevant CDRs. These template variable regionsare in a form compatable with the expression vectors used, that is,within HindIII-BamHI fragments, that also include promotor regions,signal peptide and intron DNA (FIGS. 3 and 4). For the NEWM VH version,the template is M13VHPCR1 (Orlandi et al, above, and section 8.3 below).A derivative of this template, containing KOL FRs and irrelevant CDRs,was used to generate the KOL coding region. A derivative of M13VKPCR1(Orlandi et al. above) was used in the creation of the HuVK vector. Theresulting vectors were termed M13NMHuVH, M13KLHuVH and M13HuVK. TheHindIII-BamHI fragments containing the humanized MN-14 variable regionDNAs were transferred from these M13 vectors to the expression vectorsessentially as described for the construction of the chimeric MN-14expression vectors in Example 8.1.

The NEWM FR is described in Poljak et al. Biochemistry 16: 3412-20(1977). Construction of a hMN-14 with an affinity for CEA comparable tothat of its murine counterpart was achieved in a stepwise approach.Production of the chimeric antibody provided a useful control whenassessing the binding of the humanized versions. The human NEWM VH andREI VK frameworks were initially chosen as the basis for reshaping theantibody as they are known to be tolerated in man. MN-14 residues Phe27,Asp28 and Thr30 were retained because, although not part of the Kabat'shypervariable region, CDR1 residues 31-35, those amino acids are part ofthe CDR1 structural loop (Chothia et al., J. Mol. Biol. 176: 901-917(1987)). In addition, the following residues were also selected forincorporation into the humanized VH for the following reasons: Ala24,this residue contacts CDR1; Arg71, the side chain of this residue pokesthrough the center of the domain to interact with CDRs 1 and 2;substitution of the smaller Val may alter the conformation of these CDRloops; and, Ser94, the majority of antibodies have Arg in this positionwhere it is thought to interact with an Asp residue on CDR3, and theinclusion of the Arg of NEWM could create an unwanted interaction withthe murine CDR. Other changes were made to this version (NMHuVH) inthree areas corresponding to regions which had been proved important inother reshaped molecules. These changes were: (i) Gln77Phe78Ser79 toThrLeuTyr (NMHuVhHTLY, SEQ. ID NO. 9) (ii) Ser82Thr83Ala84Ala85 toLysArgSerGlu (NMHuVhHKRSE, SEQ. ID NO. 10) (iii)Arg66Val67Thr68Met69Leu70 to LysPhelleValSer (NMHuVhKFIVS, SEQ. ID NO.11)

The alignment of the different versions of the NEWM VH frameworks (SEQ.ID NOS. 5 and 8-11) with the MN-14 VH (SEQ. ID NO. 2) is shown in FIGS.6A, 6B, and 6C. Each of these versions has been paired with the sameHuVK. The inclusion of either the TLY or KFIVS motifs gave about atwo-fold improvement.

There are 2 differences from the NEWN framework sequences given in Kabatet. al (1987) above: S107 to T and L108 to T. Kabat lists residue 1 asPCA and residues 5 and 6 as E or Q.

B. KOL Based Humanization.

In parallel to the use of NEWM VH, we have also reshaped the human KOLVH. KOL VH is described in Schmidt et al., Z. physiol. Chem, 364:713-747 (1987). The FR used for humanizing the antibody is as given inKabat. The original version of the KOL-based VH contained the MN-14CDRs, as defined in terms of residue variability (Kabat et al., above)and three additional murine residues. As with the NEWM VH, two of thesesubstitutions were made because the actual peptide CDR1 structural loop,which extends from the β-sheet framework, consists of residues 25 to 32(Chothia et al. 1987, above). Changes at positions 28 and 30 allowedthis loop to be transplanted as a whole from the murine antibody. MN-14residue 94 was included because the Arg residue of the KOL VH isinvolved in a salt bridge with Asp 110 and, like with the NEWM VH, itwas felt that retention of Arg 94 might perturb the MN-14 CDR3structure. The side-chain of residue 94 may also interact with residuesof CDR1. Other changes made to this basis MN-14 KOLHuVH (SEQ. ID NO. 12)were as follows: (i) Ser24 to Ala24 and Val48Ala49 to IleGly (KLHuVhAIG,SEQ. ID NO. 13). (ii) Ser24 to Ala24, Val48Ala49 to IleGly and Ser74 toAla (KLHuVhAIGA, SEQ. ID NO. 14). (iii) Ser 24 to Ala24, VAl48Ala49 toIleGly, Ser74 to Ala and Phe79 to Tyr (KLHuVhAIGAY, SEQ. ID NO. 15).Mutation Rationale

Ala24—The loop of CDR1 is anchored by the penetration of the side chainof residue 29 into the framework. Residue 24 is one of those with whichit interacts (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)).

Ile48Gly49—Although both these residues are adjacent to the CDR2hypervariable region they are far removed from the actual structuralloop. Both residues are completely buried (Padlan, Mol. Immunolog. 28:489-498, 1991) and it was considered possible that these would effectbinding via their packing interaction.

Ala74—This residue is part of the fourth loop found at the VHantigen-binding surface and its side chain is almost completely exposedto solvent (Padlan, 1991 above). Direct interaction of this residue withantigen could be envisaged.

Tyr79—Like residue 74, this residue is close to the antigen-binding siteand could effect binding.

The alignment of the different versions of KOL VH frameworks (SEQ. IDNOS. 7 and 12-15) with NEWM based versions (SEQ. ID NOS. 5 and 8-11) andthe murine MN14 (SEQ. ID NO. 2) is shown in FIGS. 6A, 6B, and 6C.

The DNA sequences and translation products of MN14HuVH and MN14HuVL areshown in FIGS. 7 and 8, 0respectively (SEQ. ID NOS. 16-19,respectively).

The humanized KLHuVH varients, such as antibody KLHuVHAIGA/HuVK, werepurified and tested in a blocking assay carried out as follows.Antibodies were added at the indicated concentrations together withHRP-labeled MN-14 to a final volume of 0.1 ml. Following 30 mins. ofincubation at 37₁C, and washing to remove unbound antibodies, therelative affinities of the antibodies were determined from the remainingbound peroxidase activity. A shown by the assay data of FIG. 9, theactivity of the “reshaped” (i.e., CDR-engrafted on FR) humanizedantibody was similar to that of chimeric and murine positive controls.

Results obtained using supernatant fluids from cells secretingKLHuVHAIG/HuVK and KLHuVHAIGAY/HuVK antibodies suggests that these haveblocking activities that are similar to that of the KLHuVHAIGA/HuVKantibody.

C. REI Based Humanization

REI VL is described in Epp et al., Eur. J. Biochem., 45: 513-24 (1974).No change4s were made in REI framework to improve binding. The changesfrom the sequences given in Kabat et al. (1987) are: M4 to L; T39 to K:Y71 to F; L104 to V; Q105 to E: and T107 to K. These changes preexistedin the template used when grafting the MN-14 CDRs and were not madespecifically to improve binding. The M4 to L change incorporates arestriction site but the remaining differences eliminate unusualresidues in the REI framework. A similar framework has been referred toas a consensus of huma kappa subgroup I by Foote et al., J. Mol. Biol.224: 487-9 (1992).

EXAMPLE 9 Expression of MN-14 CDR-Grafted Humanized Antibodies

Cells that were stably transformed for expression of the MN-14CDR-grafted human antibodies were selected in the manner described aboveand cloned out to establish individual producer lines. Each of the lineswas assayed to determine production of the correct antibody and toassess the efficiency of production. The antibody class was determinedand the anti-CEA binding affinity assessed.

The best producers were further characterized for the overall amount ofthe antibody produced and, for the best of these, sequences wereobtained from the mRNA to insure that the mutation has not occurred inthe antibody genes during transfection, integration, propagation orselection.

EXAMPLE 10 Purification of MN-14 CDR-Grafted Humanized AntibodiesExpressed in Cell Culture

The best producer lines of the MN-14 CDR-grafted human antibody werecultured, the growth medium collected and filtered through a 0.2 micronmembrane. The antibody was then purified by protein A chromatographyfollowed by other conventional purification steps such as ion exchangeand size exclusion chromatography. The cells were pelleted and from thesupernatant by conventional centrifugation. The antibody was purifiedfrom the supernatant fluid as described above.

EXAMPLE 11 Uses of Humanized MN-14 Monoclonal Antibody in Diagnoses

A. Animal Studies

The biodistribution of labeled humanized MN-14 IgG in nude mice bearinghuman colon cancer was determined. For radiolocalization studies, at 4-5weeks female athymic mice (nu/nu, Harlan, Indianapolis, Ind.) were givens.c. 0.2 ml of a 10% suspension of LS174T human colon adenocarcinomaprepared from a xenograft serially propagated in an athymic mouse(Sharkey et al., Cancer Res., 50: 828-34 (1990)). After waiting 2 weeksfor tumor development, the mice were injected i.v. with 20 μCi (about 2μg) of ¹³¹-labelled humanized MN-14 monoclonal antibodies. Groups of 4-5mice were sacrificed at intervals thereafter, and radioactivitylocalized in tissues according to Sharkey et al., above. The data ofTable 2 show the % injected dose/g tissue and tumor:nontumor ratios.

The results show excellent tumor accretion of the antibody, with maximumaccretion occuring within 2 days. Blood clearance of the hMN-14 antibodywas more rapid than the parental mMN-14 antibody. In addition, there washigher uptake of hMN-14 by the spleen than there was of mMN-14,reflecting the fact that the former antibody is “foreign” to the mouse.Tumor:nontumor ratios were excellent. These results demonstrate that theinventive hMN-14 mAb is capable of targeting CEA-producing tumors. TABLE2 Time Post-Injection ¹³¹I-hMN-14 IgG Tissue 4 hour 1 day 2 days 5 days7 days 14 days Percent Injected Dose Per Gram Tissue (N = 4 to 5animals) LS174T 11.8 ± 2.9  18.1 ± 14.9 32.6 ± 17.2 30.2 ± 13.4 10.6 ±15.2 11.6 ± 5.6  Weight 0.31 ± 0.07 0.37 ± 0.02 0.27 ± 0.08 0.31 ± 0.9 0.3 ± 0.2 0.40 ± 0.07 Liver 10.8 ± 2.1  6.0 ± 3.3 2.8 ± 0.5 0.8 ± 0.40.5 ± 0.7 0.08 ± 0.05 Spleen 17.0 ± 4.8  10.5 ± 8.8  4.9 ± 0.4 1.3 ± 0.60.6 ± 0.8 0.14 ± 0.09 Kidney 7.0 ± 0.8 3.1 ± 0.7 2.3 ± 0.9 0.9 ± 0.4 0.4± 0.6 0.07 ± 0.04 Lungs 8.7 ± 0.4 3.7 ± 1.4 3.7 ± 1.4 1.4 ± 0.6 0.6 ±1.0 0.11 ± 0.06 Blood 15.4 ± 6.6  5.5 ± 4.7 6.8 ± 4.1 2.3 ± 1.2 0.9 ±1.8 0.14 ± 0.13 Tumor (LS174T)/Nontumor Ratios (N = 4 to 5 amimals)Liver 1.1 ± 0.3 5.0 ± 5.0 11.1 ± 4.7  39.5 ± 7.5  24.6 ± 4.8  160 ± 28 Spleen 0.7 ± 0.3 4.2 ± 4.6 6.6 ± 3.4 24.7 ± 7.8  15.8 ± 4.4  91 ± 21Kidney 1.7 ± 0.5 5.3 ± 3.5 13.3 ± 3.6  36.1 ± 4.0  38.4 ± 12.9 173 ± 41 Lungs 1.4 ± 0.4 4.2 ± 2.2 8.2 ± 2.1 22.3 ± 2.1  25.4 ± 7.3  112 ± 15 Blood 0.9 ± 0.5 3.7 ± 0.9 5.4 ± 1.4 14.2 ± 2.9  26.0 ± 12.9 111 ± 35 

B. Clinical Studies with ¹³¹I-Labeled Humanized MN-14 IgG

Patients were entered into an Institutional Review Board-approvedprotocol at the Center for Molecular Medicine and Immunology, Newark,N.J. for a pilot investigation of the targeting and pharmacokineticbehavior of the humanized MN-14 IgG. In the case the results of whichare shown in FIG. 12, the male patient had colorectal cancer that hadmetastasized to the liver. He was injected i.v. with ¹³¹I-hMN-14 IgG (8mCi, 0.6 mg antibody) and images were taken over a six day period. Thepatient was subsequently injected with an identical dose of mMN-14 IgG.The images shown in FIG. 12 shaw the anterior abdominal view about 140 hafter each injection. The images are adjusted to exactly the sameintensity so that they are directly comparable. The results indicatethat the humanized antibody is taken up by the CEA-producing tumor aswell as the parental murine antibody. These experiments establish thepractical utility of diagnosing human CEA-producing colon cancers withthe inventive humanized MN-14 mAb.

1. A method for treating a patient comprising administering a humanizedClass III, anti-CEA, monoclonal antibody (mAb) to said patient in aneffective amount for treatment.
 2. The method of claim 1, wherein theconstant regions of said mAb are from a human IgG1 antibody.
 3. Themethod of claim 1, wherein said mAb comprises acomplementarity-determining region (CDR) selected from the groupconsisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ ID NO:21),QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD (SEQ IDNO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination thereof, whereinsaid mAb is unreactive with meconium antigen by enzyme immunoassay. 4.The method of claim 3, wherein said mAb retains the binding specificityof a parental murine Class III, anti-CEA mAb which comprises said CDRs.5. The method of claim 1, wherein said treatment comprises administeringsaid mAb to patients with a CEA-producing cancer.
 6. The method of claim5, wherein said CEA-producing cancer is selected from the groupconsisting of colon cancer, breast cancer and lung cancer.
 7. The methodof claim 1, wherein said mAb further comprises framework regions (FRs)of light and heavy chain variable regions from a human antibody.
 8. Themethod of claim 1, wherein said mAb further comprises framework regions(FRs) in the light and heavy chain variable regions, wherein each FR isseparately a FR of a human antibody.
 9. The method of claim 8, whereineach of said light and heavy chain variable regions chains comprise FRsfrom at least two human antibodies.
 10. The method of claim 7, whereinthe light chain variable region comprises a framework region (FR)selected from the group consisting of DIQLTQSPSSLSASVGDRVTITC (SEQ IDNO:26); WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29) or acombination thereof; and the heavy chain variable region comprises aframework region (FR) selected from the group consisting ofEVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30), EVQLVESGGGVVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ ID NO:33);RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ IDNO:39); and a combination thereof; and wherein C may be in thesulfhydryl or disulfide form.
 11. The method of claim 10, wherein thelight chain and heavy chain variable regions comprise FRs comprising:FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2 comprisesWYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or D)RFSGS(G or V)(SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4 comprises FGQGTKVEIK; (SEQID NO:29) FRH1 comprises EVQLVESGGGVVQPG RSLRLSCSSSGFDFT, (SEQ ID NO:30)or EVQLVESGGG VVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprisesWVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3 comprises RFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) orRFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprisesWGQGTPVTVSS; (SEQ ID NO:39)

and wherein C may be in the sulfhydryl or disulfide form.
 12. A methodfor treating a patient comprising administering a conjugate to saidpatient in an effective amount for treatment, wherein said conjugatecomprises: a therapeutic agent bound to a humanized Class III, anti-CEA,monoclonal antibody (mAb) or a fragment thereof, wherein said mAb orfragment thereof comprises a complementarity-determining region (CDR)selected from the group consisting of KASQDVGTSVA (SEQ ID NO:20),WTSTRHT (SEQ ID NO:21), QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23),EIHPDSSTINYAPSLKD (SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and acombination thereof, wherein said mAb is unreactive with meconiumantigen by enzyme immunoassay.
 13. The method of claim 12, wherein saidmAb retains the binding specificity of a parental murine Class III,anti-CEA mAb which comprises said CDRs.
 14. The method of claim 12,wherein said therapeutic agent comprises a cytotoxic agent or animmunomodulator.
 15. The method of claim 14, wherein said cytotoxicagent comprises doxorubicin, methotrexate, taxol, ricin A or aradionuclide.
 16. The method of claim 15, wherein said radionuclidecomprises ¹³¹I.
 17. The method of claim 12, further comprisingadministering a second therapeutic agent to said patient.
 18. The methodof claim 17, wherein said second therapeutic agent is doxorubicin,methotrexate, taxol, ricin A, or a radionuclide.
 19. The method of claim14, wherein said cytotoxic agent is a radionuclide, and said methodfurther comprises administering a second therapeutic agent or performinga treatment regimen to said patient.
 20. The method of claim 19, whereinsaid treatment regimen is a bone marrow reinfusion.
 21. The method ofclaim 19, wherein said radionuclide is ¹³¹I.
 22. The method of claim 12,wherein said mAb retains the binding specificity of a parental murineClass III, anti-CEA mAb which comprises said CDRs.
 23. The method ofclaim 12, wherein said treatment comprises administering said conjugateto a patient with a CEA-producing cancer.
 24. The method of claim 23,wherein said CEA-producing cancer is selected from the group consistingof colon cancer, breast cancer and lung cancer.
 25. The method of claim12, said mAb or fragment thereof further comprises framework regions(FRs) of light and heavy chain variable regions from a human antibody.26. The method of claim 12, wherein said mAb further comprises frameworkregions (FRs) in the light and heavy chain variable regions, whereineach FR is separately a FR of a human antibody.
 27. The method of claim26, wherein each of said light and heavy chain variable regions chainscomprise FRs from at least two human antibodies.
 28. The method of claim25, wherein the light chain variable region comprises a framework region(FR) selected from the group consisting of DIQLTQSPSSLSASVGDRVTITC (SEQID NO:26); WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29) and acombination thereof; and the heavy chain variable region comprises aframework region (FR) selected from the group consisting ofEVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30), EVQLVESGGGVVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ ID NO:33);RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ IDNO:39); and a combination thereof; and wherein C may be in thesulfhydryl or disulfide form.
 29. The method of claim 28, wherein thelight chain and heavy chain variable regions comprise FRs comprising:FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2 comprisesWYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or D)RFSGS(G or V)(SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4 comprises FGQGTKVEIK; (SEQID NO:29) FRH1 comprises EVQLVESGGGVVQPG RSLRLSCSSSGFDFT, (SEQ ID NO:30)or EVQLVESGGG VVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprisesWVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3 comprises RFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) orRFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4 comprisesWGQGTPVTVSS; (SEQ ID NO:39)

and wherein C may be in the sulfhydryl or disulfide form.
 30. A methodfor diagnosing a patient comprising administering a conjugate to saidpatient in an effective amount for diagnosis, wherein said conjugatecomprises: a diagnostic agent bound to a humanized Class III, anti-CEA,monoclonal antibody (mAb) or a fragment thereof, wherein said mAb orfragment thereof comprises a complementarity-determining region (CDR)selected from the group consisting of KASQDVGTSVA (SEQ ID NO:20),WTSTRHT (SEQ ID NO:21), QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23),EIHPDSSTINYAPSLKD (SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and acombination thereof, wherein said mAb is unreactive with meconiumantigen by enzyme immunoassay.
 31. The method of claim 30, wherein saidmAb retains the binding specificity of a parental murine Class III,anti-CEA mAb which comprises said CDRs.
 32. The method of claim 30,wherein said diagnostic agent comprises an imaging agent.
 33. The methodof claim 32, wherein said imaging agent is a radionuclide or a metalchelator complexed radionuclide.
 34. The method of claim 33, whereinsaid radionuclide is iodine, yttrium or technetium.
 35. The method ofclaim 34, wherein said radionuclide is ¹³¹I.
 36. The method of claim 30,wherein said diagnosis comprises administering said conjugate to apatient with a CEA-producing cancer.
 37. The method of claim 36, whereinsaid CEA-producing cancer is selected from the group consisting of coloncancer, breast cancer and lung cancer.
 38. The method of claim 30, saidmAb or fragment thereof further comprises framework regions (FRs) oflight and heavy chain variable regions from a human antibody.
 39. Themethod of claim 30, wherein said mAb further comprises framework regions(FRs) in the light and heavy chain variable regions, wherein each FR isseparately a FR of a human antibody.
 40. The method of claim 39, whereineach of said light and heavy chain variable regions chains comprise FRsfrom at least two human antibodies.
 41. The method of claim 38, whereinthe light chain variable region comprises a framework region (FR)selected from the group consisting of DIQLTQSPSSLSASVGDRVTITC (SEQ IDNO:26); WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29) and acombination thereof; and the heavy chain variable region comprises aframework region (FR) selected from the group consisting ofEVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30), EVQLVESGGGVVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ ID NO:33);RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ IDNO:39); and a combination thereof; and wherein C may be in thesulfhydryl or disulfide form.
 42. The method of claim 41, wherein thelight chain and heavy chain variable regions comprise FRs comprising:FRL1 comprises DIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2 comprisesWYQQKPGKAPKLLIY; (SEQ ID NO:27) FRL3 comprises GVP(S or D)RFSGS(G or V)(SEQ ID NO:28) SGTDFTFTISSLQPEDIATYYC; FRL4 comprises FGQGTKVEIK; (SEQID NO:29) FRH1 comprises EVQLVESGGGVVQPG RSLRLSCSSSGFDFT, (SEQ ID NO:30)or EVQLVESGGG VVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprisesWVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3 comprises RFTIS (SEQ ID NO:36)RDNSKNTLFLQMDSLRPEDTGVYFCAS, or RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQID NO:37) and FRH4 comprises WGQGTPVTVSS; (SEQ ID NO:39)

and wherein C may be in the sulfhydryl or disulfide form.
 43. A ClassIII, anti-CEA monoclonal antibody (mAb) or antigen-binding fragmentthereof comprising a complementarity-determining region (CDR) selectedfrom the group consisting of KASQDVGTSVA (SEQ ID NO:20), WTSTRHT (SEQ IDNO:21), QQYSLYRS (SEQ ID NO:22), TYWMS (SEQ.ID NO:23), EIHPDSSTINYAPSLKD(SEQ ID NO:24), LYFGFPWFAY (SEQ ID NO:25), and a combination thereof,wherein said mAb is unreactive with meconium antigen by enzymeimmunoassay.
 44. The mAb or fragment thereof of claim 43, wherein saidmAb retains the binding specificity of a parental murine Class III,anti-CEA mAb which comprises said CDRs.
 45. The mAb or fragment thereofof claim 44, wherein the light chain variable region of said mAb orfragment thereof comprises CDRL1 comprising KASQDVGTSVA (SEQ ID NO:20),CDRL2 comprising WTSTRHT (SEQ ID NO:21), and CDRL3 comprising QQYSLYRS(SEQ ID NO:22), and the heavy chain variable region of said mAb orfragment thereof comprises CDRH1 comprising TYWMS (SEQ.ID NO:23), CDRH2comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24) and CDRH3 comprisingLYFGFPWFAY (SEQ ID NO:25).
 46. The mAb of claim 45, said mAb or fragmentthereof is a humanized Class III, anti-CEA mAb and further comprisesframework regions (FRs) of light and heavy chain variable regions from ahuman antibody.
 47. The mAb of claim 46, wherein: (a) the light chainvariable regions are characterized by the formula:FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein each FR is a differentframework region of a human antibody, and FRL1 comprises a region ofabout 23 amino acids that occurs naturally in the FRL1 of a humanantibody; FRL2 comprises a region of about 15 amino acids that occursnaturally in the FRL2 of a human antibody; FRL3 comprises a region ofabout 32 amino acids that occurs naturally in the FRL3 of a humanantibody; FRL4 comprises a region of about 10 amino acids that occursnaturally in the FRL4 of a human antibody; and (b) the heavy chainvariable regions are characterized by the formula:FRH1—CDRH1—FRH2-CDRH2—FRH3-CDRH3—FRH4, wherein each FR is a differentframework region of a human antibody, and FRH1 comprises a region of28-32 amino acids that occurs naturally in the FRH1 of a human antibody;FRH2 comprises a region of 12-16 amino acids that occurs naturally inthe FRH2 of a human antibody; FRH3 comprises a region of 30-34 aminoacids that occurs naturally in the FRH3 of a human antibody; and FRH4comprises a region of 9-13 amino acids that occurs naturally in the FRH4of a human antibody.
 48. The mAb of claim 47, wherein FRL1 comprisesDIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2 comprises WYQQKPGKAPKLLIY;(SEQ ID NO:27) FRL3 comprises GVP(S or D)RFSGS(G or V) (SEQ ID NO:28)SGTDFTFTISSLQPEDIATYYC; FRL4 comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1comprises EVQLVESGGG VQPGRSLRLSCSSSGFDFT, (SEQ ID NO:30) orEVQLVESGGGVVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprisesWVRQAPGKGLEWVA; (SEQ ID NO:33) FRH3 comprisesRFTISRDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ ID NO:36) orRFTISRDNAKNTLFLQMDSLRPEDTGVYFC AS; (SEQ ID NO:37) and FRH4 comprisesWGQGTPVTVSS; (SEQ ID NO:39) and wherein C may be in the sulfhydryl ordisulfide form.


49. A humanized Class III, anti-CEA mAb comprising framework regions(FRs) of light and heavy chain variable regions from a human antibody,wherein the light chain variable region comprises a framework region(FR) selected from the group consisting of DIQLTQSPSSLSASVGDRVTITC (SEQID NO:26); WYQQKPGKAPKLLIY (SEQ ID NO:27); GVP(S or D)RFSGS(G or V)SGTDFTFTISSLQPEDIATYYC (SEQ ID NO:28); FGQGTKVEIK (SEQ ID NO:29) and acombination thereof; and the heavy chain variable region comprises aframework region (FR) selected from the group consisting ofEVQLVESGGGVVQPG RSLRLSCSSSGFDFT (SEQ ID NO:30), EVQLVESGGGVVQPGRSLRLSCSASGFDFT (SEQ ID NO:31); WVRQAPGKGLEWVA (SEQ ID NO:33);RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:36),RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS (SEQ ID NO:37); WGQGTPVTVSS (SEQ IDNO:39); and a combination thereof; and wherein C may be in thesulfhydryl or disulfide form; and wherein said mAb is unreactive withmeconium antigen by enzyme immunoassay and unreactive with normaltissue.
 50. The mAb of claim 49, wherein the light chain and heavy chainvariable regions comprise FRs comprising: FRL1 comprisesDIQLTQSPSSLSASVGDRVTITC; (SEQ ID NO:26) FRL2 comprises WYQQKPGKAPKLLIY;(SEQ ID NO:27) FRL3 comprises GVP(S or D)RFSGS(G or V) (SEQ ID NO:28)SGTDFTFTISSLQPEDIATYYC; FRL4 comprises FGQGTKVEIK; (SEQ ID NO:29) FRH1comprises EVQLVESGGGVVQPG RSLRLSCSSSGFDFT, (SEQ ID NO:30) or EVQLVESGGGVVQPGRSLRLSCSASGFDFT; (SEQ ID NO:31) FRH2 comprises WVRQAPGKGLEWVA; (SEQID NO:33) FRH3 comprises RFTIS RDNSKNTLFLQMDSLRPEDTGVYFCAS, (SEQ IDNO:36) or RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS; (SEQ ID NO:37) and FRH4comprises WGQGTPVTVSS; (SEQ ID NO:39)

and wherein C may be in the sulfhydryl or disulfide form.
 51. Anisolated polynucleotide comprising a nucleic acid sequence encoding themAb or fragment thereof of claim
 43. 52. An expression vector comprisingthe isolated polynucleotide of claim
 51. 53. A transformed cellcomprising the isolated polynucleotide of claim
 51. 54. The transformedcell of claim 53, wherein said cell is a mammalian cell.
 55. A method ofproducing a Class III, anti-CEA mAb comprising (a) transforming a cellwith the isolated polynucleotide of claim 51; and (b) expressing saidpolynucleotide by culturing said cell; and (c) producing said mAb.
 56. Acomplementarity determining region (CDR) comprising KASQDVGTSVA (SEQ IDNO:20).
 57. A complementarity determining region (CDR) comprisingWTSTRHT (SEQ ID NO:21).
 58. A complementarity determining region (CDR)comprising QQYSLYRS (SEQ ID NO:22).
 59. A complementarity determiningregion (CDR) comprising TYWMS (SEQ.ID NO:23).
 60. A complementaritydetermining region (CDR) comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24).61. A complementarity determining region (CDR) comprising LYFGFPWFAY(SEQ ID NO:25).